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Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

Bottom Line: As a result, there is much interest in understanding the dynamics of autophagy in a variety of situations.As such, these assays do not measure changes in endogenous cargo degradation which is the ultimate end-point of the autophagy process.We consider therefore that this assay will prove to be a valuable resource for investigations in which autophagy is considered important and is believed to be modulated.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

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Enhanced-mitophagy can detect genetic loss of autophagy. ATG7flox/flox MEFs expressing YFP-Parkin together with either a retrovirus expressing Cre recombinase or control virus were treated with 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were analyzed by Western blotting was undertaken using the MitoProfile Membrane integrity WB Antibody Cocktail, an anti-LC3B antibody and an anti-actin antibody as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.
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f0025: Enhanced-mitophagy can detect genetic loss of autophagy. ATG7flox/flox MEFs expressing YFP-Parkin together with either a retrovirus expressing Cre recombinase or control virus were treated with 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were analyzed by Western blotting was undertaken using the MitoProfile Membrane integrity WB Antibody Cocktail, an anti-LC3B antibody and an anti-actin antibody as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.

Mentions: We lastly decided to test if enhanced-mitophagy can detect genetic loss of autophagy. To do this we isolated fibroblasts from E13.5 mouse embryos which were homozygous for a floxed allele of the essential autophagy gene Atg7. Following isolation and immortalization, this floxed allele was recombined in vitro by infection with a retrovirus expressing Cre recombinase, with an ‘empty’ retroviral vector being used as control (Fig. 5). Subsequent infection of these cells with YFP-Parkin resulted in virtually complete ablation of CVA, Core 1, Porin, cyclophilin D and cytochrome c when control cells were treated with antimycin A and oligomycin (Fig. 5). By contrast, in YFP-Parkin-expressing Atg7- cells while loss of Porin and cytochrome c occurs in a similar manner to what was observed in control cells, the depletion of CVa, Core 1 and cyclophilin D was markedly impaired due to loss of autophagy (Fig. 5). We therefore conclude that enhanced-mitophagy can be used to detect genetic loss of autophagy and that CVa, Core 1 and cyclophilin D are once again the reliable mitochondrial proteins to monitor when using this assay.


Using enhanced-mitophagy to measure autophagic flux.

Baudot AD, Haller M, Mrschtik M, Tait SW, Ryan KM - Methods (2014)

Enhanced-mitophagy can detect genetic loss of autophagy. ATG7flox/flox MEFs expressing YFP-Parkin together with either a retrovirus expressing Cre recombinase or control virus were treated with 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were analyzed by Western blotting was undertaken using the MitoProfile Membrane integrity WB Antibody Cocktail, an anti-LC3B antibody and an anti-actin antibody as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358839&req=5

f0025: Enhanced-mitophagy can detect genetic loss of autophagy. ATG7flox/flox MEFs expressing YFP-Parkin together with either a retrovirus expressing Cre recombinase or control virus were treated with 1 μM antimycin A + 1 μM oligomycin for 24 h. Lysates were analyzed by Western blotting was undertaken using the MitoProfile Membrane integrity WB Antibody Cocktail, an anti-LC3B antibody and an anti-actin antibody as a loading control. The immunoblots shown are representative of what was seen in three independent experiments.
Mentions: We lastly decided to test if enhanced-mitophagy can detect genetic loss of autophagy. To do this we isolated fibroblasts from E13.5 mouse embryos which were homozygous for a floxed allele of the essential autophagy gene Atg7. Following isolation and immortalization, this floxed allele was recombined in vitro by infection with a retrovirus expressing Cre recombinase, with an ‘empty’ retroviral vector being used as control (Fig. 5). Subsequent infection of these cells with YFP-Parkin resulted in virtually complete ablation of CVA, Core 1, Porin, cyclophilin D and cytochrome c when control cells were treated with antimycin A and oligomycin (Fig. 5). By contrast, in YFP-Parkin-expressing Atg7- cells while loss of Porin and cytochrome c occurs in a similar manner to what was observed in control cells, the depletion of CVa, Core 1 and cyclophilin D was markedly impaired due to loss of autophagy (Fig. 5). We therefore conclude that enhanced-mitophagy can be used to detect genetic loss of autophagy and that CVa, Core 1 and cyclophilin D are once again the reliable mitochondrial proteins to monitor when using this assay.

Bottom Line: As a result, there is much interest in understanding the dynamics of autophagy in a variety of situations.As such, these assays do not measure changes in endogenous cargo degradation which is the ultimate end-point of the autophagy process.We consider therefore that this assay will prove to be a valuable resource for investigations in which autophagy is considered important and is believed to be modulated.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Beatson Institute, Garscube Estate, Switchback Rd, Glasgow G61 1BD, UK.

Show MeSH
Related in: MedlinePlus